The present invention will now be described with particular reference to biomedical applications. However, it is to be understood that the present invention has applications in any area that requires increased resistance to bacterial and fungal colonization.
The colonization by bacteria of devices used for human health care and/or improvement of quality of life poses serious problems and causes adverse reactions that are detrimental to the viability and useful service life of the device. Examples of devices that are colonized by bacteria comprise implantable biomedical devices such as urinary catheters, percutaneous access catheters, stents, as well as non-implantable devices such as contact lenses, contact lens storage cases, and the like. Once attached to a surface, bacteria are much more resistant to antibiotics and other bacteriostatic and bacteriocidal agents and can proliferate, inducing adverse reactions in the host environment.
Accordingly, much work has focused on the prevention of bacterial colonization. One solution is to coat catheters with a thin layer of silver metal, which releases silver ions that act as antibacterial agents. Another solution is the surface immobilization of quaternary amine compounds (Dziabo U.S. Pat. No. 5,515,117) which are known to be antibacterial agents.
The provision of silver coatings is not practical or economic in many applications. There is also clinical evidence that silver coatings do not provide adequate effectiveness in all desirable circumstances. Quaternary amine compounds likewise possess shortcomings in that they can induce cell toxicity with host cells, which adversely affects the continued viability of fully functioning tissues adjacent to the biomedical implant. While the cytotoxicity of quaternary amine compounds is relatively mild, it is possible to observe in in vitro cell culture experiments that the shape and biological functions of cells in contact with such compounds are substantially affected.
Furanone compounds have been reported to be effective agents against bacterial proliferation and to have antifungal properties (see for example Reichelt and Borowitzka (1984) Hydrobiologia 116: 158-168 and International Patent Application Nos. PCT/AU99/00284 and PCT/AU96/00167, the disclosures of which are incorporated herein by reference). They are thought to act by interfering with bacterial properties that are regulated by acylated homoserine lactones, (AHLs) and two component phosphorelay signal transduction systems. These are fundamental regulatory agents which are widespread in bacteria, including human pathogens (see for example International Patent Application Nos. PCT/AU96/00167 and PCT/AU00/01553, the disclosures of which is incorporated herein by reference).
The AHL regulatory systems in bacteria are one type of signal transduction systems which regulate intercellular activity in response to environmental conditions and extracellular signal molecules. This system was first discovered in the bioluminescent mane bacteria Vibrio harveyi and V. fischeri where it is used to control expression of bioluminescence. In principle, the system is comprised of two proteins—LuxR and LuxI. The LuxI enzyme is encoded by a luxI gene and produces a related family of signal molecules known as the acylated homoserine lactones (AHLs). These signal molecules bind to the LuxR regulator which is then activated and serves both as a positive regulator for the structural genes which encode the enzymes responsible for bioluminescence, and as a positive regulator for the luxI gene itself. The entire system is amplified via a process of auto induction. Additional molecules serve as regulators of the LuxR-LuxI system.
While initially discovered for bioluminescent bacteria, this regulatory system has now been found in numerous other microorganisms, and is involved in a wide variety of bacterial activities (Pesci and Iglewski, 1999, In Cell-cell signaling in bacteria. Dunny and Winans (eds), ASM Press, Washington D.C., Stevens and Greenberg, 1999 In Cell-cell signaling in bacteria. Dunny and Winans (eds), ASM Press, Washington D.C., Pierson et al. 1998, Annu. Rev. Phytopathol. 36:207-225). These activities include, but are not restricted to exoenzyme production in the plant pathogen Erwinia carotovora and exoenzyme and virulence factor production in Pseudomonas aeruginosa, the causative agent of cystic fibrosis, and Ti plasmid transfer from Agrobacterium tumefaciens to plants. In all instances acylated homoserine lactone, or homoserine lactone-like compounds are the regulatory autoinducers.
Two-component phosphorelay signal transduction systems represent another mechanism, which is distinct from the AHL system described above, by which bacteria sense and respond to their environment (see PCT/AU00/01553). Two-component transduction systems play important roles in the growth and maintenance and functionality of many different microorganisms. Examples include, but not are limited to, regulation of the production of exopolysaccharides and virulence factors; the regulation of motility, swarming, attachment and biofilm formation; and maintenance of viability.
Since these regulatory systems are widespread among bacteria and because they control processes leading to bacterial invasion of host organisms, it is likely that other organisms will have evolved defense mechanisms against these systems.
Natural furanones and their synthetic analogues have been shown to inhibit bacterial adhesion (PCT/AU96/00167). The presumed mode of action of interfering with the regulation of AHL and two component phosphorelay systems entails that the compounds should be capable of diffusing into and through the bacterial cell in order to reach the target site. As a result, soluble, low-molecular weight furanones have been used to date as antibacterial agents.
However, surprisingly, we have found that furanone compounds immobilized onto polymeric substrate surfaces by stable covalent bonds still maintain antibacterial activity, in preventing bacterial proliferation on that substrate material. This surprising finding is at odds with the presumed intracellular action of furanone compounds as AHL mimics, and their ability to interfere with signal transduction through the two-component phosphorelay systems and cannot be explained at present with a well-supported mechanistic model.